The function and mechanisms of action of ghrelin and obestatin in ovarian cancer

In this study, we have demonstrated that the preproghrelin derived hormones, ghrelin and obestatin, may play a role in ovarian cancer. Ghrelin and obestatin stimulated an increase in cell migration in ovarian cancer cell lines and may play a role in cancer progression. Ovarian cancer is the leading cause of death among gynaecological cancers and is the sixth most common cause of cancer-related deaths in women in developed countries. As ovarian cancer is difficult to diagnose at a low tumour grade, two thirds of ovarian cancers are not diagnosed until the late stages of cancer development resulting in a poor prognosis for the patient. As a result, current treatment methods are limited and not ideal. There is an urgent need for improved diagnostic markers, as well better therapeutic approaches and adjunctive therapies for this disease. Ghrelin has a number of important physiological effects, including roles in appetite regulation and the stimulation of growth hormone release. It is also involved in regulating the immune, cardiovascular and reproductive systems and regulates sleep, memory and anxiety, and energy metabolism. Over the last decade, the ghrelin axis, (which includes the hormones ghrelin and obestatin and their receptors), has been implicated in the pathogenesis of many human diseases and it may t may also play an important role in the development of cancer. Ghrelin is a 28 amino acid peptide hormone that exists in two forms. Acyl ghrelin (usually referred to as ghrelin), has a unique n-octanoic acid post-translational modification (which is catalysed by ghrelin O-acyltransferase, GOAT), and desacyl ghrelin, which is a non-octanoylated form. Octanoylated ghrelin acts through the growth hormone secretagogue receptor type 1a (GHSR1a). GHSR1b, an alternatively spliced isoform of GHSR, is C-terminally truncated and does not bind ghrelin. Ghrelin has been implicated in the pathophysiology of a number of diseases Obestatin is a 23 amino acid, C-terminally amidated peptide which is derived from preproghrelin. Although GPR39 was originally thought to be the obestatin receptor this has been disproven, and its receptor remains unknown. Obestatin may have as diverse range of roles as ghrelin. Obestatin improves memory, inhibits thirst and anxiety, increases pancreatic juice secretion and has cardioprotective effects. Obestatin also has been shown to regulate cell proliferation, differentiation and apoptosis in some cell types. Prior to this study, little was known regarding the functions and mechanisms of action ghrelin and obestatin in ovarian cancer. In this study it was demonstrated that the full length ghrelin, GHSR1b and GOAT mRNA transcripts were expressed in all of the ovarian-derived cell lines examined (SKOV3, OV-MZ-6 and hOSE 17.1), however, these cell lines did not express GHSR1a. Ovarian cancer tissue of varying stages and normal ovarian tissue expressed the coding region for ghrelin, obestatin, and GOAT, but not GHSR1a, or GHSR1b. No correlations between cancer grade and the level of expression of these transcripts were observed. This study demonstrated for the first time that both ghrelin and obestatin increase cell migration in ovarian cancer cell lines. Treatment with ghrelin (for 72 hours) significantly increased cell migration in the SKOV3 and OV-MZ-6 ovarian cancer cell lines. Ghrelin (100 nM) stimulated cell migration in the SKOV3 (2.64 +/- 1.08 fold, p <0.05) and OV-MZ-6 (1.65 +/- 0.31 fold, p <0.05) ovarian cancer cell lines, but not in the representative normal cell line hOSE 17.1. This increase in migration was not accompanied by an increase in cell invasion through Matrigel. In contrast to other cancer types, ghrelin had no effect on proliferation. Ghrelin treatment (10nM) significantly decreased attachment of the SKOV3 ovarian cancer cell line to collagen IV (24.7 +/- 10.0 %, p <0.05), however, there were no changes in attachment to the other extracellular matrix molecules (ECM) tested (fibronectin, vitronectin and collagen I), and there were no changes in attachment to any of the ECM molecules in the OV-MZ-6 or hOSE 17.1 cell lines. It is, therefore, unclear if ghrelin plays a role in cell attachment in ovarian cancer. As ghrelin has previously been demonstrated to signal through the ERK1/2 pathway in cancer, we investigated ERK1/2 signalling in ovarian cancer cell lines. In the SKOV3 ovarian cancer cell line, a reduction in ERK1/2 phosphorylation (0.58 fold +/- 0.23, p <0.05) in response to 100 nM ghrelin treatment was observed, while no significant change in ERK1/2 signalling was seen in the OV-MZ-6 cell line with treatment. This suggests that this pathway is unlikely to be involved in mediating the increased migration seen in the ovarian cancer cell lines with ghrelin treatment. In this study ovarian cancer tissue of varying stages and normal ovarian tissue expressed the coding region for obestatin, however, no correlation between cancer grade and level of obestatin transcript expression was observed. In the ovarian-derived cell lines studied (SKOV3, OV-MZ-6 and hOSE 17.1) it was demonstrated that the full length preproghrelin mRNA transcripts were expressed in all cell lines, suggesting they have the ability to produce mature obestatin. This is the first study to demonstrate that obestatin stimulates cell migration and cell invasion. Obestatin induced a significant increase in migration in the SKOV3 ovarian cancer cell line with 10 nM (2.80 +/- 0.52 fold, p <0.05) and 100 nM treatments (3.12 +/- 0.68 fold, p <0.05) and in the OV-MZ-6 cancer cell line with 10 nM (2.04 +/- 0.10 fold, p <0.01) and 100 nM treatments (2.00 +/- 0.37 fold, p <0.05). Obestatin treatment did no affect cell migration in the hOSE 17.1normal ovarian epithelial cell line. Obestatin treatment (100 nM) also stimulated a significant increase in cell invasion in the OV-MZ-6 ovarian cancer cell line (1.45 fold +/- 0.13, p <0.05) and in the hOSE17.1 normal ovarian cell line cells (1.40 fold +/- 0.04 and 1.55 fold +/- 0.05 respectively, p <0.01) with 10 nM and 100 nM treatments. Obestatin treatment did not stimulate cell invasion in the SKOV3 ovarian cancer cell line. This lack of obestatin-stimulated invasion in the SKOV3 cell line may be a cell line specific result. In this study, obestatin did not stimulate cell proliferation in the ovarian cell lines and it has previously been shown to have no effect on cell proliferation in the BON-1 pancreatic neuroendocrine and GC rat somatotroph tumour cell lines. In contrast, obestatin has been shown to affect cell proliferation in gastric and thyroid cancer cell lines, and in some normal cell lines. Obestatin also had no effect on attachment of any of the cell lines to any of the ECM components tested (fibronectin, vitronectin, collagen I and collagen IV). The mechanism of action of obestatin was investigated further using a two dimensional-difference in gel electrophoresis (2D-DIGE) proteomic approach. After treatment with obestating (0, 10 and 100 nM), SKOV3 ovarian cancer and hOSE 17.1 normal ovarian cell lines were collected and 2D-DIGE analysis and mass spectrometry were performed to identify proteins that were differentially expressed in response to treatment. Twenty-six differentially expressed proteins were identified and analysed using Ingenuity Pathway Analysis (IPA). This linked 16 of these proteins in a network. The analysis suggested that the ERK1/2 MAPK pathway was a major mediator of obestatin action. ERK1/2 has previously been shown to be associated with obestatin-stimulated cell proliferation and with the anti-apoptotic effects of obestatin. Activation of the ERK1/2 signalling pathway by obestatin was, therefore, investigated in the SKOV3 and OV-MZ-6 ovarian cancer cell lines using anti-active antibodies and Western immunoblots. Obestatin treatment significantly decreased ERK1/2 phosphorylation at higher obestatin concentrations in both the SKOV3 (100 nM and 1000 nM) and OV-MZ-6 (1000 nM) cell lines compared to the untreated controls. Currently, very little is known about obestatin signalling in cancer. This thesis has demonstrated for the first time that the ghrelin axis may play a role in ovarian cancer migration. Ghrelin and obestatin increased cell migration in ovarian cancer cell lines, indicating that they may be a useful target for therapies that reduce ovarian cancer progression. Further studies investigating the role of the ghrelin axis using in vivo ovarian cancer metastasis models are warranted.

Ghrelin O-acyltransferase (GOAT) is expressed in prostate cancer tissues and cell lines and expression is differentially regulated in vitro by ghrelin

Background Ghrelin is a 28 amino acid peptide hormone that is expressed in the stomach and a range of peripheral tissues, where it frequently acts as an autocrine/paracrine growth factor. Ghrelin is modified by a unique acylation required for it to activate its cognate receptor, the growth hormone secretagogue receptor (GHSR), which mediates many of the actions of ghrelin. Recently, the enzyme responsible for adding the fatty acid residue (octanoyl/acyl group) to the third amino acid of ghrelin, GOAT (ghrelin O-acyltransferase), was identified. Methods We used cell culture, quantitative real-time reverse transcription (RT)-PCR and immunohistochemistry to demonstrate the expression of GOAT in prostate cancer cell lines and tissues from patients. Real-time RT-PCR was used to demonstrate the expression of prohormone convertase (PC)1/3, PC2 and furin in prostate cancer cell lines. Prostate-derived cell lines were treated with ghrelin and desacyl ghrelin and the effect on GOAT expression was measured using quantitative RT-PCR. Results We have demonstrated that GOAT mRNA and protein are expressed in the normal prostate and human prostate cancer tissue samples. The RWPE-1 and RWPE-2 normal prostate-derived cell lines and the LNCaP, DU145, and PC3 prostate cancer cell lines express GOAT and at least one other enzyme that is necessary to produce mature, acylated ghrelin from proghrelin (PC1/3, PC2 or furin). Finally, ghrelin, but not desacyl ghrelin (unacylated ghrelin), can directly regulate the expression of GOAT in the RWPE-1 normal prostate derived cell line and the PC3 prostate cancer cell line. Ghrelin treatment (100nM) for 6 hours significantly decreased GOAT mRNA expression two-fold (P < 0.05) in the PC3 prostate cancer cell line, however, ghrelin did not regulate GOAT expression in the DU145 and LNCaP prostate cancer cell lines. Conclusions This study demonstrates that GOAT is expressed in prostate cancer specimens and cell lines. Ghrelin regulates GOAT expression, however, this is likely to be cell-type specific. The expression of GOAT in prostate cancer supports the hypothesis that the ghrelin axis has autocrine/paracrine roles. We propose that the RWPE-1 prostate cell line and the PC3 prostate cancer cell line may be useful for investigating GOAT regulation and function.

Structural characterization of glycerophospholipids by combinations of ozone- and collision-induced dissociation mass spectrometry: The next step towards "top-down" lipidomics

The complete structural elucidation of complex lipids, including glycerophospholipids, using only mass spectrometry represents a major challenge to contemporary analytical technologies. Here, we demonstrate that product ions arising from the collision-induced dissociation (CID) of the [M + Na] + adduct ions of phospholipids can be isolated and subjected to subsequent gas-phase ozonolysis-known as ozone-induced dissociation (OzID)-in a linear ion-trap mass spectrometer. The resulting CID/OzID experiment yields abundant product ions that are characteristic of the acyl substitution on the glycerol backbone (i.e., sn-position). This approach is shown to differentiate sn-positional isomers, such as the regioisomeric phosphatidylcholine pair of PC 16:0/18:1 and PC 18:1/16:0. Importantly, CID/OzID provides a sensitive diagnostic for the existence of an isomeric mixture in a given sample. This is of very high value for the analysis of tissue extracts since CID/OzID analyses can reveal changes in the relative abundance of isomeric constituents even within different tissues from the same animal. Finally, we demonstrate the ability to assign carbon-carbon double bond positions to individual acyl chains at specific backbone positions by adding subsequent CID and/or OzID steps to the workflow and that this can be achieved in a single step using a hybrid triple quadrupole-linear ion trap mass spectrometer. This unique approach represents the most complete and specific structural analysis of lipids by mass spectrometry demonstrated to date and is a significant step towards comprehensive top-down lipidomics. This journal is © The Royal Society of Chemistry 2014. Grant Number ARC/DP0986628, ARC/FT110100249, ARC/LP110200648

Gas-Phase Transformation of Phosphatidylcholine Cations to Structurally Informative Anions via Ion/Ion Chemistry

Gas-phase transformation of synthetic phosphatidylcholine (PC) monocations to structurally informative anions is demonstrated via ion/ion reactions with doubly deprotonated 1,4-phenylenedipropionic acid (PDPA). Two synthetic PC isomers, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC16:0/18:1) and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (PC18:1/16:0), were subjected to this ion/ion chemistry. The product of the ion/ion reaction is a negatively charged complex, \[PC + PDPA - H](-). Collisional activation of the long-lived complex causes transfer of a proton and methyl cation to PDPA, generating \[PC - CH3](-). Subsequent collisional activation of the demethylated PC anions produces abundant fatty acid carboxylate anions and low-abundance acyl neutral losses as free acids and ketenes. Product ion spectra of \[PC - CH3](-) suggest favorable cleavage at the sn-2 position over the sn-1 due to distinct differences in the relative abundances. In contrast, collisional activation of PC cations is absent of abundant fatty acid chain-related product ions and typically indicates only the lipid class via formation of the phosphocholine cation. A solution phase method to produce the gas-phase adducted PC anion is also demonstrated. Product ion spectra derived from the solution phase method are similar to the results generated via ion/ion chemistry. This work demonstrates a gas-phase means to increase structural characterization of phosphatidylcholines via ion/ion chemistry. Grant Number ARC/CE0561607, ARC/DP120102922

Formation and Fragmentation of Unsaturated Fatty Acid M-2H+Na (-) Ions: Stabilized Carbanions for Charge-Directed Fragmentation

Fatty acids are long-chain carboxylic acids that readily produce \[M - H](-) ions upon negative ion electrospray ionization (ESI) and cationic complexes with alkali, alkaline earth, and transition metals in positive ion ESI. In contrast, only one anionic monomeric fatty acid-metal ion complex has been reported in the literature, namely \[M - 2H + (FeCl)-Cl-II](-). In this manuscript, we present two methods to form anionic unsaturated fatty acid-sodium ion complexes (i.e., \[M - 2H + Na](-)). We find that these ions may be generated efficiently by two distinct methods: (1) negative ion ESI of a methanolic solution containing the fatty acid and sodium fluoride forming an \[M - H + NaF](-) ion. Subsequent collision-induced dissociation (CID) results in the desired \[M - 2H + Na](-) ion via the neutral loss of HF. (2) Direct formation of the \[M - 2H + Na](-) ion by negative ion ESI of a methanolic solution containing the fatty acid and sodium hydroxide or bicarbonate. In addition to deprotonation of the carboxylic acid moiety, formation of \[M - 2H + Na](-) ions requires the removal of a proton from the fatty acid acyl chain. We propose that this deprotonation occurs at the bis-allylic position(s) of polyunsaturated fatty acids resulting in the formation of a resonance-stabilized carbanion. This proposal is supported by ab initio calculations, which reveal that removal of a proton from the bis-allylic position, followed by neutral loss of HX (where X = F- and -OH), is the lowest energy dissociation pathway.

Differentiation of complex lipid isomers by radical-directed dissociation mass spectrometry

Contemporary lipidomics protocols are dependent on conventional tandem mass spectrometry for lipid identification. This approach is extremely powerful for determining lipid class and identifying the number of carbons and the degree of unsaturation of any acyl-chain substituents. Such analyses are however, blind to isomeric variants arising from different carbon carbon bonding motifs within these chains including double bond position, chain branching, and cyclic structures. This limitation arises from the fact that conventional, low energy collision-induced dissociation of even-electron lipid ions does not give rise to product ions from intrachain fragmentation of the fatty acyl moieties. To overcome this limitation, we have applied radical-directed dissociation (RDD) to the study of lipids for the first time. In this approach, bifunctional molecules that contain a photocaged radical initiator and a lipid-adducting group, such as 4-iodoaniline and 4-iodobenzoic acid, are used to form noncovalent complexes (i.e., adduct ions) with a lipid during electrospray ionization. Laser irradiation of these complexes at UV wavelengths (266 nm) cleaves the carbon iodine bond to liberate a highly reactive phenyl radical. Subsequent activation of the nascent radical ions results in RDD with significant intrachain fragmentation of acyl moieties. This approach provides diagnostic fragments that are associated with the double bond position and the positions of chain branching in glycerophospholipids, sphingomyelins and triacylglycerols and thus can be used to differentiate isomeric lipids differing only in such motifs. RDD is demonstrated for well-defined lipid standards and also reveals lipid structural diversity in olive oil and human very-low density lipoprotein.

A comparison of patient matched meibum and tear lipidomes

Purpose. To quantify the molecular lipid composition of patient-matched tear and meibum samples and compare tear and meibum lipid molecular profiles. Methods. Lipids were extracted from tears and meibum by bi-phasic methods using 10:3 tertbutyl methyl ether:methanol, washed with aqueous ammonium acetate, and analyzed by chipbased nanoelectrospray ionization tandem mass spectrometry. Targeted precursor ion and neutral loss scans identified individual molecular lipids and quantification was obtained by comparison to internal standards in each lipid class. Results. Two hundred and thirty-six lipid species were identified and quantified from nine lipid classes comprised of cholesterol esters, wax esters, (O-acyl)-x-hydroxy fatty acids, triacylglycerols, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine. With the exception of phospholipids, lipid molecular profiles were strikingly similar between tears and meibum. Conclusions. Comparisons between tears and meibum indicate that meibum is likely to supply the majority of lipids in the tear film lipid layer. However, the observed higher mole ratio of phospholipid in tears shows that analysis of meibum alone does not provide a complete understanding of the tear film lipid composition.

Analysis of unsaturated lipids by ozone-induced dissociation

Recent developments in analytical technologies have driven significant advances in lipid science. The sensitivity and selectivity of modern mass spectrometers can now provide for the detection and even quantification of many hundreds of lipids in a single analysis. In parallel, increasing evidence from structural biology suggests that a detailed knowledge of lipid molecular structure including carbon-carbon double bond position, stereochemistry and acyl chain regiochemistry is required to fully appreciate the biochemical role(s) of individual lipids. Here we review the capabilities and limitations of tandem mass spectrometry to provide this level of structural specificity in the analysis of lipids present in complex biological extracts. In particular, we focus on the capabilities of a novel technology termed ozone-induced dissociation to identify the position (s) of double bonds in unsaturated lipids and discuss its possible role in efforts to develop workflows that provide for complete structure elucidation of lipids by mass spectrometry alone: so-called top-down lipidomics. This article is part of a Special Issue entitled: Lipodomics and Imaging Mass Spectrom. (C) 2011 Elsevier B.V. All rights reserved.

Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin induced dormant ring stages of Plasmodium falciparum

Artemisinin (ART) based combination therapy (ACT) is used as the first line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART induced ring stage dormancy and recovery has been implicated as possible cause of recrudescence; however, little is known about the characteristics of dormant parasites including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA) induced dormancy and recovery. Transcription analysis showed an immediate down regulation for 10 genes following exposure to DHA, but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, were also maintained. Additions of inhibitors for biotin acetyl CoA carbozylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively following DHA treatment. Our results demonstrate most metabolic pathways are down regulated in DHA induced dormant parasites. In contrast fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment.

The ghrelin axis in the skeletal microenvironment

This project characterised the bone microarchitecture of adult mice lacking the hormone, acyl ghrelin, by high resolution micro-computed tomography; and investigated the expression of the ghrelin axis in cells of human and mouse fetal cartilage. This thesis highlights for the first time the physiological role of the ghrelin axis in the bone microenvironment of aged mice. Furthermore it improves our understanding of the complex expression patterns of the ghrelin axis in cartilage cells of human and mouse fetal skeletons.

A proteomic view into infection of greyback canegrubs (Dermolepida albohirtum) by Metarhizium anisopliae

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.

Characterization of the tail-specific protease (Tsp) from Legionella

Bacterial tail-specific proteases (Tsps) have been attributed a wide variety of functions including intracellular virulence, cell wall morphology, proteolytic signal cascades and stress response. This study tested the hypothesis that Tsp has a key function for the transmissive form of Legionella pneumophila. A tsp mutant was generated in Legionella pneumophila 130b and the characteristics of this strain and the isogenic wild-type were examined using a range of growth and proteomic analyses. Recombinant Tsp protein was also produced and analyzed. The L. pneumophila tsp mutant showed no defect in growth on rich media or during thermo-osmotic stress conditions. In addition, no defects in cellular morphology were observed when the cells were examined using transmission electron microscopy. Purified recombinant Tsp was found to be an active protease with a narrow substrate range. Proteome analysis using iTRAQ (5% coverage of the proteome) found that, of those proteins detected, only 5 had different levels in the tsp mutant compared to the wild type. ACP (Acyl Carrier Protein), which has a key role for Legionella differentiation to the infectious form, was reduced in the tsp mutant; however, tsp(-) was able to infect and replicate inside macrophages to the same extent as the wild type. Combined, these data demonstrate that Tsp is a protease but is not essential for Legionella growth or cell infection. Thus, Tsp may have functional redundancy in Legionella.

5,7-di-N-acetyl-acinetaminic acid: A novel non-2-ulosonic acid found in the capsule of an Acinetobacter baumannii isolate

An Acinetobacter baumannii global clone 1 (GC1) isolate was found to carry a novel capsule biosynthesis gene cluster, designated KL12. KL12 contains genes predicted to be involved in the synthesis of simple sugars, as well as ones for N-acetyl-l-fucosamine (l-FucpNAc) and N-acetyl-d-fucosamine (d-FucpNAc). It also contains a module of 10 genes, 6 of which are required for 5,7-di-N-acetyl-legionaminic acid synthesis. Analysis of the composition of the capsule revealed the presence of N-acetyl-d-galactosamine, l-FucpNAc and d-FucpNAc, confirming the role of fnlABC and fnr/gdr genes in the synthesis of l-FucpNAc and d-FucpNAc, respectively. A non-2-ulosonic acid, shown to be 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-altro-non-2-ulosonic acid, was also detected. This sugar has not previously been recovered from biological source, and was designated 5,7-di-N-acetyl-acinetaminic acid (Aci5Ac7Ac). Proteins encoded by novel genes, named aciABCD, were predicted to be involved in the conversion of 5,7-di-N-acetyl-legionaminic acid to Aci5Ac7Ac. A pathway for 5,7-di-N-acetyl-8-epilegionaminic acid biosynthesis was also proposed. In available A. baumannii genomes, genes for the synthesis of 5,7-di-N-acetyl-acinetaminic acid were only detected in two closely related capsule gene clusters, KL12 and KL13, which differ only in the wzy gene. KL12 and KL13 are carried by isolates belonging to clinically important clonal groups, GC1, GC2 and ST25. Genes for the synthesis of N-acyl derivatives of legionaminic acid were also found in 10 further A. baumannii capsule gene clusters, and three carried additional genes for production of 5,7-di-N-acetyl-8-epilegionaminic acid.

Comparative analysis and distribution of Omega-3 lcPUFA Biosynthesis genes in marine molluscs

Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the required enzymes for the synthesis of lcPUFAs.